GD3 ganglioside is a promising target for immunotherapy in patients with melanoma due to: a) abundant expression on virtually all melanoma, b) restricted distribution on normal tissue, and c) the observation that treatment of melanoma patients with mouse monoclonal antibodies (MAb) against GD3 has resulted in major clinical responses without significant side effects. Attempts to immunize patients against GD3, and provide active immunity, using melanoma cells, lysates or purified GD3 have been unsuccessful due to the low immunogenicity of GD3. To overcome this, we have developed a mouse anti-idiotypic (anti-id) MAb that mimics GD3 ganglioside. Rabbits immunized with this anti-id MAb, designated BEC2, develop IgG specifically against GD3. The broad, long-term objectives of this application are to explore the ability of anti-id MAb to induce immunity against non-protein tumor antigens and to determine how to optimize this form of vaccine. The specific aim of this proposal is to determine which portion of BEC2 is required to mimic GD3. This is important because: a) anti-id MAb mimicking non-protein tumor antigens are rare and the mechanism of how a protein mimics a glycolipid is not understood, b) this will permit further improvements of the vaccine including bioengineering a chimeric molecule, and c) this may shed light on how other non-protein tumor antigens can be made immunogenic. The BEC2 hybridoma produces two light chains and a heavy chain which have already been sequenced. The possibility exists that a second heavy chain is also produced. Initial experiments will focus on determining which heavy and light chain is responsible for mimicking GD3. Subsequent studies will more finely map the regions within the heavy and light chains responsible for mimicking GD3.